Field
The present disclosure is drawn to in vitro methods of producing platelets from stem cells for clinical use.
Description of the Related Art
Each year, millions of patients in the United States are affected by various blood disorders and diseases, such as thrombocytopenia (low platelet number), that require multiple treatments of platelet transfusions. Although more than 10 million platelet donations are made annually (all of which come from volunteer donors) the demand continues to increase at a greater rate than the supply. The process of obtaining platelets, however, is not only lengthy and costly, but it is further limited by a shelf life of only a few days. This short window of usability means that many donated platelet units are discarded before having an opportunity to serve the patients in need of these valuable products.
Platelets are tiny blood cells that perform the vital and highly specialized function of blood clotting. Almost a trillion platelets circulate in the average person's blood, and the turnover is such that the entire platelet population is replaced every 10 days. This represents a tremendous amount of ongoing platelet production. Platelets have a highly organized cytoskeleton and intracellular stores of over 300 proteins, which they secrete at sites of blood vessel injury. Platelets also play a role in inflammation, blood vessel growth, and tumor metastasis.
Platelets (thrombocytes) are small, irregularly shaped clear cell fragments 2-3 μm in diameter, which are derived from fragmentation of precursor megakaryocytes. Megakaryocytes are derived from hematopoietic stem cell precursor cells in the bone marrow These multipotent stem cells live in the marrow sinusoids and are capable of producing all types of blood cells depending on the signals they receive. The primary signal for megakaryocyte production is thrombopoietin (TPO). TPO induces differentiation of progenitor cells in the bone marrow towards a final megakaryocyte phenotype. The megakaryocyte develops through the following lineage: CFU-ME (pluripotential hemopoietic stem cell or hemocytoblast)→megakaryoblast→promegakaryocyte→megakaryocyte. The cell eventually reaches megakaryoblast stage and loses its ability to divide. However, it is still able to replicate its DNA and continue development, becoming polyploid. The cytoplasm continues to expand and the DNA complement can increase to greater than 64 N.
Once the cell has completed differentiation and becomes a mature megakaryocyte, it begins the process of producing platelets. TPO plays a role in inducing the megakaryocyte to form small proto-platelet processes. Platelets are held within these internal membranes within the cytoplasm of the megakaryocytes. There are two proposed mechanisms for platelet release. In one scenario, these proto-platelet processes break up explosively to become platelets. Alternatively, the cell may form platelet ribbons into blood vessels. The ribbons are formed via pseudopodia and they are able to continuously emit platelets into circulation. In either scenario, each of these proto-platelet processes can give rise to 2000-5000 new platelets upon breakup. Overall, more than 75% of these newly-produced platelets will remain in circulation while the remainder will be sequestered by the spleen.
Thrombocytopenia, a major medical problem affecting millions of patients per year in the US, can result in spontaneous bleeding and is treated using various methods to increase platelet production. The condition can result from malignancy and chemotherapy, immune disorders such as immune thrombocytopenia (ITP), infection, and major surgery. There are also a large number of inherited platelet defects that cause excessive bleeding. All of these serious medical conditions may require treatment at some point with life-saving platelet transfusions
There has been much interest in the possibility of using stem cells to produce platelets in the laboratory for clinical use. Stem cells are undifferentiated cells in early stage of development and capable of giving rise to more cells of the same type or differentiating into a diverse range of cell lineages. The main different types of stem cells are human embryonic stem cells (HeSC), induced pluripotent stem cells (IPSC), and hematopoietic stem cells (HSC).
HeSC are pluripotent stem cells derived from the inner cell mass of an early-stage embryo and are capable of differentiating into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. These cells are capable of differentiating into all kinds of cells in the human body. IPSC are a type of pluripotent stem cell artificially derived from a mature cell. Typically, adult somatic cells are induced to become pluripotent by activating specific genes of immaturity in these cells. Hematopoietic stem cells are progenitor cells that circulate in the blood and reside in the bone marrow and have the potential to give rise to all hematopoietic cells. Hematopoietic stem cells can be acquired from the bone marrow, from peripheral blood with apheresis machines, or from umbilical cord or placenta after birth.
Culture systems have been described for differentiating stem cells into the various types of blood cells. There were expectations that stem cells, such as hematopoietic, HeSC and IPSC, could be used to generate blood cells for clinical use. Despite the successful production of functional platelets in the laboratory, reported yields have been far too low for clinical use and the field is currently at a technical impasse. As an example, one unit of umbilical cord blood may contain about 106 (one million) CD34+ cells. One million CD34+ cells yield up to 107 platelets under current optimal conditions. In contrast, a typical platelet transfusion delivers about 3×1011 platelets. Thus, an increase in efficiency is needed to provide a transfusion of cultured platelets to equal the number of platelets from one unit of umbilical cord blood.